CN105037364A - Pyrimidine replacing purine compound as kinase inhibitor - Google Patents

Pyrimidine replacing purine compound as kinase inhibitor Download PDF

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CN105037364A
CN105037364A CN201510344718.7A CN201510344718A CN105037364A CN 105037364 A CN105037364 A CN 105037364A CN 201510344718 A CN201510344718 A CN 201510344718A CN 105037364 A CN105037364 A CN 105037364A
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cancer
disease
compound
syndrome
formula
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CN105037364B (en
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陈迪忠
M·威廉姆斯
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SBio Pte Ltd
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SBio Pte Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • C07D473/26—Heterocyclic compounds containing purine ring systems with an oxygen, sulphur, or nitrogen atom directly attached in position 2 or 6, but not in both
    • C07D473/32—Nitrogen atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • C07D473/40—Heterocyclic compounds containing purine ring systems with halogen atoms or perhalogeno-alkyl radicals directly attached in position 2 or 6
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
    • C07F5/02—Boron compounds
    • C07F5/025—Boronic and borinic acid compounds

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The invention relates to a purine compound capable of being used as a kinase inhibitor. The compound adopts a structure referring to the description. The invention also relates to salt acceptable in pharmacy of the purine compound.

Description

As the purine compound that the pyrimidine of kinase inhibitor replaces
Patent application of the present invention is international application no is PCT/SG2009/000124, international filing date is on April 3rd, 2009, the application number entering National Phase in China is 200980159441.3, and denomination of invention is the divisional application of the application for a patent for invention of " purine compound that the pyrimidine as kinase inhibitor replaces ".
Technical field
The present invention relates to 5-(9-sec.-propyl-8-methyl-2-morpholine-4-base-9H-purine-6-base)-pyrimidine-2-base amine, its preparation method, comprises pharmaceutical composition and the application of this compound in some kinase-associated conditions/illness for the treatment of of this compound.
Background technology
Verified to the research of kinase inhibitor is develop the useful fruitful field of pharmaceutically active substances.Kinases or be called phosphotransferase to be called the enzyme in Phosphorylation events, phosphate group being transferred to certain target molecules (so-called substrate) from high energy donor molecule (such as ATP).A class maximum in kinases is protein kinase, and they act on specified protein and regulate it active.
Because kinase inhibitor can be used as pharmaceutical active compounds, carry out large quantity research has suitable activity to these target spots compound with exploitation.At cancer field, the two kinds of kinases that can be used as the potential target spot of therapeutic compound aroused attention comprise mTOR and PI3.A research example in this field is shown in PCT/SG2008/000379, the application discloses many compounds mTOR and PI3 to kinase activity.
Expection suppresses the compound of mTOR and PI3K can provide potent antiproliferative, angiogenesis inhibitor and anti-tumor activity, because these compounds can act on the multiple points in PI3K/Akt/mTOR path simultaneously.At present, this type of inhibitor many are just carrying out clinical study first (e.g., BEZ235, XL765, GDC0941, PX866, SF1126).
In the process finding suitable drug candidate, whether a kind of compound of final decision is that suitable drug candidate needs to consider many factors.Therefore, in the process evaluating the potential compound being used for exploitation further, except the main inhibit activities of compound itself, also need to consider many other factorses.In evaluation procedure, those skilled in the art are conceived to " drug-like properties " of molecule, comprise the solubleness (if they are insoluble, they are not too suitable as Subsequent pharmacological usually) of evaluation such as its activity to target spot interested, compound of interest, compound in vivo with external metabolic stability, compound to factors such as the issuable side effects of human body.The application identifies other Compound Phase ratio with this field, the compound that drug-like properties significantly improves.
Summary of the invention
The invention provides the compound of formula (I):
Or its pharmacy acceptable salt.
Except the compound of formula I, the embodiment disclosed also relates to this compound pharmacy acceptable salt, pharmaceutically acceptable N-oxide compound, pharmaceutically acceptable prodrug and pharmaceutical active metabolite, and the pharmacy acceptable salt of this metabolite.
The invention still further relates to pharmaceutical composition, comprise the compounds of this invention and pharmaceutically acceptable carrier, thinner or vehicle.
Another aspect of the present invention provides a kind of arrestin kinase whose method, described protein kinase is selected from lower group: serine/threonine protein kitase or its fragment or mixture or its functional equivalent and PI3 kinases or its fragment or mixture or its functional equivalent, and the method comprises makes protein kinase or its fragment or mixture or its functional equivalent and/or its cofactor contact with the compounds of this invention of significant quantity.
This compound disclosed can directly with act solely on kinase molecule or its mixture or fragment, to suppress biological activity.However, it should be understood that, described compound can also act on the cofactor participated in Phosphorylation events at least partly.Known kinases cofactor comprises ionic species (as zinc and calcium), lipid (as phosphatidy serine) and DG.
In some embodiments, protein kinase is serine/threonine protein kitase or its fragment or mixture or its functional equivalent.In some embodiments, described serine/threonine protein kitase or its fragment or mixture are mTOR protein kinase or its fragment or its mixture or its functional equivalent.In some embodiments, described serine/threonine protein kitase is mTORC1 or its fragment or mixture or its functional equivalent.In some embodiments, described serine/threonine protein kitase is mTORC2 or its fragment or mixture or its functional equivalent.
In some embodiments, protein kinase is PI3 kinases or its fragment or its mixture or its functional equivalent.In some embodiments, described PI3 kinases or its fragment or its mixture or its functional equivalent are I class PI3K or its fragment or its mixture or its functional equivalent.
In a kind of embodiment of the method making one or more protein kinases contact with described compound, comprise the Mammals giving described compound to contain one or more protein kinases.
In another aspect of the present invention, there is provided the compounds of this invention to suppress the application of one or more protein kinases, described protein kinase is selected from lower group: serine/threonine protein kitase or its fragment or mixture or its functional equivalent and PI3 kinases or its fragment or mixture or its functional equivalent.
In some embodiments, protein kinase is serine/threonine protein kitase or its fragment or mixture or its functional equivalent.In some embodiments, described serine/threonine protein kitase or its fragment or mixture are mTOR protein kinase or its fragment or its mixture or its functional equivalent.In some embodiments, described serine/threonine protein kitase is mTORC1 or its fragment or mixture or its functional equivalent.In some embodiments, described serine/threonine protein kitase is mTORC2 or its fragment or mixture or its functional equivalent.
In some embodiments, protein kinase is PI3 kinases or its fragment or its mixture or its functional equivalent.In some embodiments, described PI3 kinases or its fragment or its mixture or its functional equivalent are I class PI3K or its fragment or its mixture or its functional equivalent.
In another aspect of the present invention, a kind of method for the treatment of or prevention mammalian diseases is provided, wherein, suppress one or more protein kinases being selected from serine/threonine protein kitase or its fragment or mixture or its functional equivalent and PI3 kinases or its fragment or mixture or its functional equivalent can prevent, suppress or improve symptom or the symptom of described disease, described method comprises the compound of the present invention giving to treat significant quantity.
In some embodiments, protein kinase is serine/threonine protein kitase or its fragment or mixture or its functional equivalent.In some embodiments, described serine/threonine protein kitase or its fragment or mixture are mTOR protein kinase or its fragment or its mixture or its functional equivalent.In some embodiments, described serine/threonine protein kitase is mTORC1 or its fragment or mixture or its functional equivalent.In some embodiments, described serine/threonine protein kitase is mTORC2 or its fragment or mixture or its functional equivalent.
In some embodiments, protein kinase is PI3 kinases or its fragment or its mixture or its functional equivalent.In some embodiments, described PI3 kinases or its fragment or its mixture or its functional equivalent are I class PI3K or its fragment or its mixture or its functional equivalent.
In some embodiments, described illness is cancer.In some embodiments, cancer is selected from lower group: hematologic cancer, as myeloproliferative disease (idiopathic myelofibrosis, polycythemia vera, essential thrombocythemia, chronic myeloid leukemia), myeloid metaplasia, chronic myelomonocytic leukemia, acute lymphoblastic leukemia, acute erythroblastic leukemia, Huo Qijin and non-Hodgkin lymphoma, B cell lymphoma, acute T-cell leukemia, myelodysplastic syndrome, plasmocyte obstacle, hairy cell, Kaposi sarcoma, lymphoma and hyperproliferation illness are as psoriasis and restenosis, gynecological cancer, as mammary cancer, ovarian cancer, cervical cancer, vagina and vulva cancer, endometrial hyperplasia, gastrointestinal cancer, as colorectal carcinoma, polyp, liver cancer, cancer of the stomach, carcinoma of the pancreas, carcinoma of gallbladder, urinary tract cancer, as prostate cancer, kidney and kidney portion cancer, bladder cancer, urethral carcinoma, penile cancer, skin carcinoma, as melanoma, cerebral tumor, as glioblastoma, neuroblastoma, astrocytoma, ependymoma (ependynoma), brain stem glioma, medulloblastoma, meningioma (menigiomas), astrocytoma, oligodendroglioma, incidence cancer, as nasopharyngeal carcinoma, laryngocarcinoma, respiratory cancer, as lung cancer (NSCLC and SCLC), mesothelioma, illness in eye, as retinoblastoma, musculoskeletal disease, as osteosarcoma, flesh and bone tumor, squamous cell cancer and fibroma.In other embodiments, compound of the present invention can be used for treatment precancerosis disease (pre-cancercondition) or hyperplasia, comprising: tumorigenesis in familial adenomatous polyposis, adenomatous polyposis coli, myelodysplasia, Endometrium development exception, endometrial hyperplasia with atypia, cervical atypical hyperplasia, vagina epithelium, benign prostatic hyperplasia, larynx papilloma, photochemical and solar keratosis, seborrheic keratosis and keratoacanthoma.
In some embodiments, the described illness disease that is autoimmunization or inflammatory diseases or caused by excessive neovascularization.Following disease is comprised to a certain extent: acute disseminated encephalomyelitis owing to the autoimmunization cause of disease or the disease that relates to pathologic inflammatory and new vessel forming reactions, addison's disease, without gamma-globulin mass formed by blood stasis (agammaglobulinermia), granulopenia, allergic asthma, allergic encephalitis, rhinallergosis, alopecia areata, senile alopecia, red corpuscle occurs can not, ankylosing spondylitis, antiphospholipid antibody syndrome, aortitis syndrome, aplastic anemia, atopic dermatitis, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmunization ovaritis, balo disease, Basedow disease (Basedow'sdisease), behcet disease/syndrome, bronchial asthma, block this Mann syndrome (Castleman ' ssyndrome), celiac disease, chagas disease, chronic inflammatory demyelinating polyneuropathy, churg-Strauss syndrome (Chrug-Strausssyndrome), Ke's radicular syndrome (Coganssyndrome), keratoconus (cornicalcornea), keratoconus hickie (cornicalleukoma), coxsackie myocarditis, CREST is sick, Crohn's disease, epidermis eosinophilia, cutaneous T-cell lymphoma, diversity dermatitis erythremia, dermatomyositis, diabetic retinopathy, postmyocardial infarction syndrome, dystrophia epithelialis corneae, eczematoid dermatitis, eosinophilic fasciitis, eosinophilic gastroenteritis, epidermolysis bullosa, Evans syndrome (Evanssyndrome), FA, gestation pemphigoid, glomerulonephritis, Goodpasture's syndrome, graft versus host disease (GVH disease), Graves disease, guillain-Barre syndrome (Guillain-Barresyndrome), Hashimoto's disease, hemolytic uremic syndrome, herpetic keratitis, ichthyosis vulgaris, idiopathic interstitial pneumonia, idiopathic thrombocytopenic purpura, inflammatory bowel, kawasaki disease, keratitis, keratoconjunctivitis, Lambert-Eaton syndrome, common white pinta, lichen planus, lichen sclerosus, Lyme disease, linear IgA bullous disease (linearIgAdisease), macular degeneration, megaloblastic anemia, Meniere disease (Meniere ' sdisease), Mooren's ulcer (Mooren ' sulcer), mucha-Habermann disease, polymyositis, multiple sclerosis, myasthenia gravis, necrotizing enterocolitis, neuromyelitis optica, ocular pemphigus, ocular myoclonus syndrome, Ao Deshi thyroiditis (Ord ' sthyroiditis), paroxysmal nocturnal hemoglobinuria, Ba-Te two Cotard (Parsonnage-Turnersyndrome), pemphigus, periodontitis, pernicious anemia, pollen allergy, polyadenous autoimmune syndromes, posterior uveitis, monolobular cirrhosis, rectitis, pseudomembranous colitis, psoriasis, pulmonary emphysema, pyoderma, Reiter syndrome, reversible obstructive airway disease, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleritis, Sezary syndrome, Sjogren syndrome, subacute bacterium endocarditis, systemic lupus erythematous, Takayasu arteritis, temporal arteritis, painful ophthalmoplegia, type i diabetes, ulcerative colitis, urticaria, vernal conjunctivitis, leukodermia, Wo Ji-Vogt-Koyanagi-Harada syndrome (Vogy-Koyanagi-Haradasyndrome) and Wegener granulomatosis.In some embodiments, described illness is endometriosis.
In another aspect of the present invention, there is provided the compounds of this invention in the application of the medicine for the preparation for the treatment of Animal diseases, wherein, one or more protein kinases being selected from serine/threonine protein kitase or its fragment or mixture or its functional equivalent and PI3 kinases or its fragment or mixture or its functional equivalent are suppressed can to prevent, suppress or improve symptom or the symptom of described disease.
In another aspect of the present invention, the compounds of this invention or its pharmacy acceptable salt, N-oxide compound or the application of its prodrug in disease therapy are provided, wherein, one or more protein kinases being selected from serine/threonine protein kitase or its fragment or mixture or its functional equivalent and PI3 kinases or its fragment or mixture or its functional equivalent are suppressed can to prevent, suppress or improve symptom or the symptom of described disease.
In some embodiments, protein kinase is serine/threonine protein kitase or its fragment or mixture or its functional equivalent.In some embodiments, described serine/threonine protein kitase or its fragment or mixture are mTOR protein kinase or its fragment or its mixture or its functional equivalent.In some embodiments, described serine/threonine protein kitase is mTORC1 or its fragment or mixture or its functional equivalent.In some embodiments, described serine/threonine protein kitase is mTORC2 or its fragment or mixture or its functional equivalent.
In some embodiments, protein kinase is PI3 kinases or its fragment or its mixture or its functional equivalent.In some embodiments, described PI3 kinases or its fragment or its mixture or its functional equivalent are I class PI3K or its fragment or its mixture or its functional equivalent.
In another aspect of the present invention, provide the method for the proliferative disease of a kind of prevention or treatment target, the method comprises the compound of the present invention giving to treat significant quantity.
In another aspect of the present invention, provide the compounds of this invention in the application of the medicine for the preparation for the treatment of target proliferative disease.
In some embodiments, described illness is cancer.In some embodiments, cancer is selected from lower group: hematologic cancer, as myeloproliferative disease (idiopathic myelofibrosis, polycythemia vera, essential thrombocythemia, chronic myeloid leukemia), myeloid metaplasia, chronic myelomonocytic leukemia, acute lymphoblastic leukemia, acute erythroblastic leukemia, Huo Qijin and non-Hodgkin lymphoma, B cell lymphoma, acute T-cell leukemia, myelodysplastic syndrome, plasmocyte obstacle, hairy cell, Kaposi sarcoma, lymphoma; Gynecological cancer, as mammary cancer, ovarian cancer, cervical cancer, vagina and vulva cancer, endometrial hyperplasia; Gastrointestinal cancer, as colorectal carcinoma, polyp, liver cancer, cancer of the stomach, carcinoma of the pancreas, carcinoma of gallbladder; Urinary tract cancer, as prostate cancer, kidney and kidney portion cancer; Bladder cancer, urethral carcinoma, penile cancer; Skin carcinoma, as melanoma; Cerebral tumor, as glioblastoma, neuroblastoma, astrocytoma, ependymoma, brain stem glioma, medulloblastoma, meningioma, astrocytoma, oligodendroglioma; Incidence cancer, as nasopharyngeal carcinoma, laryngocarcinoma; Respiratory cancer, as lung cancer (NSCLC and SCLC), mesothelioma; Illness in eye, as retinoblastoma; Musculoskeletal is sick, as osteosarcoma, flesh and bone tumor; Squamous cell cancer and fibroma.In some embodiments, described illness is endometriosis.
In addition, if applicable, formula (I) should cover solvation and the nonsolvated forms of compound.Therefore, variously comprise the compound with specified structure, comprise hydration and nonhydrated form.
Other features of these characteristic sum of the present invention are as follows.
Detailed Description Of The Invention
Use multiple term known by the technical staff in this description.But multiple term will be defined for clarity.
Term " pharmacy acceptable salt " refers to the required bioactive salt of the compound retaining above qualification, comprises pharmaceutically acceptable acid salt and base addition salt.On the suitable pharmaceutical of compound shown in formula (I), acceptable acid salt can from mineral acid or organic acid preparation.The example of these mineral acids comprises hydrochloric acid, sulfuric acid and phosphoric acid.Suitable organic acid is optional from the aliphatic series of organic acid, cyclic aliphatic, aromatics, heterocyclic carboxylic acid and sulphonic acids, and the example is formic acid, acetic acid, propionic acid, succsinic acid, oxyacetic acid, glyconic acid, lactic acid, oxysuccinic acid, tartrate, citric acid, FUMARIC ACID TECH GRADE, maleic acid, alkylsulphonic acid, aryl sulfonic acid.Out of Memory about pharmacy acceptable salt is shown in " Lei Mingdun pharmaceutical science " (Remington'sPharmaceuticalSciences), 19th edition, Mack Publishing Company (MackPublishingCo.), Easton, Pennsylvania, 1995.When medicine is solid, technician should know that the compounds of this invention, medicament and salt can exist the not syncrystallization or polymorphic forms, and all these materials all should belong in the scope of the present invention and shown structural formula.
Term " treatment significant quantity " or " significant quantity " are for being enough to the consumption realizing useful or required clinical effectiveness.Significant quantity can once or several times give.Significant quantity is enough to make the process of morbid state alleviate, improve, stablize, reverse, slow down or postpone usually.
Term " function equivalent " should comprise the variant of concrete protein kinase kind described herein.Should know that kinases can have various isotype, although make the one-level of given kinase isoform, secondary, three grades or quaternary structure be different from prototype kinases, this molecule remains the biological activity of protein kinase.Isotype can produce from normal allele sudden change in crowd, the sudden change such as comprise such as aminoacid replacement, deletion, insertion, brachymemma or repeat.Term " function equivalent " also comprises the variant that transcriptional level produces.Other function equivalent comprises the kinases that posttranslational modification (such as glycosylation) is changed.
With the Compound Phase ratio of this area similar, the compounds of this invention has excellent drug-like properties, will describe in more detail below.The character prompting of these excellences, the compounds of this invention may be suitable as the material standed for of this area drug development.Observe as first time, the compounds of this invention shows the activity of quite (even if not being more excellent) in suppression two kinds of kinases interested and mTOR and PI3.All comparative compounds of the specific activity test of the compounds of this invention are stronger, and the activity of its antagonism mTOR is suitable and in the acceptable level for the treatment of use with comparative compound.
Although enzymic activity test shows, nearly all comparative compound has acceptable activity level, and the further test prompts chemical compound lot of compound can not be used as drug development based on other reason.Such as, the compounds of this invention has suitable water-soluble level (178 μMs), thus can be mixed with the pharmaceutical preparation of oral absorption, and many comparative compounds does not have acceptable solubleness.Therefore, the compounds of this invention has excellent activity and acceptable solubility characteristics simultaneously.
Have at the same time in the compound of activity and solubleness, the metabolic stability of the compounds of this invention is more excellent.The compounds of this invention has excellent stability in the research of human liver's microsome, shows its sane and relative tolerance degraded in physiological environment.On the contrary, there is other compound that is active and solubleness unstable in these researchs.Therefore, compound of the present invention has the unique combination of activity, solubleness and stability, makes it be excellent drug candidate compared to the related compound of this area, although some in these compounds have obviously structural similarity.
Compound of the present invention can suppress the activity of some protein kinase.The ability suppressing kinase activity is that the compounds of this invention directly acts solely on kinase molecule to suppress bioactive result.However, it should be understood that, described compound can also act on the described kinase whose cofactor participated in Phosphorylation events at least partly.Compound has activity to PI3 protein kinase or its fragment or mixture or its functional equivalent.Compound can to some serine/threonine kinase as mTOR or its fragment or mixture or its functional equivalent have activity.
Many modes known in the art can carry out protein kinase suppression.Such as, if need protein kinase vitro inhibition, the compounds of this invention adding appropriate amount in the kinase whose solution of purifying can contained.Requiring to suppress in the situation of kinase activity in Mammals, suppressing kinases to be usually directed to be given by this compound containing this kinase whose Mammals.
Therefore, the compounds of this invention can have various application, utilizes it to suppress the ability of the above-mentioned type protein kinase.Such as, described compound can be used for suppressing serine/threonine protein kitase.Also can be used for treatment or prevent mammiferous illness, wherein suppressing its protein kinase and/or cofactor can prevent, suppress or improve symptom or the symptom of described disease.
The compound disclosed has the ability that can be used in treating proliferative disease.The example of described disease is cancer.Expect that described compound can treat solid tumor and liquid tumors.In some embodiments, the example of the cancer can treated by the compounds of this invention comprises solid tumor and blood cancer.
As used herein, term " cancer " is that intention comprises with the generic term of uncontrolled abnormal growth of cells many illnesss that are feature.Expect that compound of the present invention can be used for the treatment of various cancer, include but not limited to: osteocarcinoma, brain and cns tumor, mammary cancer, colorectal carcinoma, internal secretion gland cancer comprises adrenocortical carcinoma, carcinoma of the pancreas, pituitary body cancer, thyroid carcinoma, Parathyroid cancer, thymic carcinoma, gastrointestinal cancer, liver cancer, the outer cancer (extrahepaticbileductcancer) of hepatic duct, stomach and intestine carcinoid tumor, bladder cancer, apparatus urogenitalis cancer, gynecological cancer, incidence cancer, leukemia, myelomatosis, blood disorder, lung cancer, lymphoma, cancer eye, skin carcinoma, soft tissue sarcoma, adult soft tissue sarcoma, Kaposi sarcoma, urinary system cancer.
Can comprise by the example of the cancer of the compounds of this invention treatment: hematologic cancer, as myeloproliferative disease (idiopathic myelofibrosis, polycythemia vera, essential thrombocythemia, chronic myeloid leukemia), myeloid metaplasia, chronic myelomonocytic leukemia, acute lymphoblastic leukemia, acute erythroblastic leukemia, Huo Qijin and non-Hodgkin lymphoma, B cell lymphoma, acute T-cell leukemia, myelodysplastic syndrome, plasmocyte obstacle, hairy cell, Kaposi sarcoma, lymphoma and hyperproliferation illness, as psoriasis and restenosis, gynecological cancer, as mammary cancer, ovarian cancer, cervical cancer, vagina and vulva cancer, endometrial hyperplasia, gastrointestinal cancer, as colorectal carcinoma, polyp, liver cancer, cancer of the stomach, carcinoma of the pancreas, carcinoma of gallbladder, urinary tract cancer, as prostate cancer, kidney and kidney portion cancer, bladder cancer, urethral carcinoma, penile cancer, skin carcinoma is as melanoma, cerebral tumor, as glioblastoma, neuroblastoma, astrocytoma, ependymoma, brain stem glioma, medulloblastoma, meningioma, astrocytoma, oligodendroglioma, incidence cancer, as nasopharyngeal carcinoma, laryngocarcinoma, respiratory cancer, as lung cancer (NSCLC and SCLC), mesothelioma, illness in eye, as retinoblastoma, musculoskeletal is sick, as osteosarcoma, flesh and bone tumor, squamous cell cancer and fibroma.Compound of the present invention is also used for the treatment of precancerosis disease or hyperplasia, comprises tumorigenesis in familial adenomatous polyposis, adenomatous polyposis coli, myelodysplasia, Endometrium development exception, endometrial hyperplasia with atypia, cervical atypical hyperplasia, vagina epithelium, benign prostatic hyperplasia, larynx papilloma, photochemical and solar keratosis, seborrheic keratosis and keratoacanthoma.
Also expect that compound of the present invention can be used for the disease for the treatment of autoimmunization or inflammatory diseases or being caused by excessive neovascularization.To a certain extent owing to the disease of the autoimmunization cause of disease, or the disease participating in Inflammation and neovascularization reaction includes but not limited to: acute disseminated encephalomyelitis, addison's disease, without gamma-globulin mass formed by blood stasis, granulopenia, allergic asthma, allergic encephalitis, rhinallergosis, alopecia areata, senile alopecia, red corpuscle occurs can not, ankylosing spondylitis, antiphospholipid antibody syndrome, aortitis syndrome, aplastic anemia, atopic dermatitis, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmunization ovaritis, balo disease, Basedow disease, behcet disease/syndrome, bronchial asthma, block this Mann syndrome, celiac disease, chagas disease, chronic inflammatory demyelinating polyneuropathy, churg-Strauss syndrome, Ke's radicular syndrome, keratoconus, keratoconus hickie, coxsackie myocarditis, CREST is sick, Crohn's disease, epidermis eosinophilia, cutaneous T-cell lymphoma, diversity dermatitis erythremia, dermatomyositis, diabetic retinopathy, postmyocardial infarction syndrome, dystrophia epithelialis corneae, eczematoid dermatitis, eosinophilic fasciitis, eosinophilic gastroenteritis, epidermolysis bullosa, Evans syndrome, FA, gestation pemphigoid, glomerulonephritis, Goodpasture's syndrome, graft versus host disease (GVH disease), Graves disease, guillain-Barre syndrome, Hashimoto's disease, hemolytic uremic syndrome, herpetic keratitis, ichthyosis vulgaris, idiopathic interstitial pneumonia, idiopathic thrombocytopenic purpura, inflammatory bowel disease, kawasaki disease, keratitis, keratoconjunctivitis, Lambert-Eaton syndrome, common white pinta, lichen planus, lichen sclerosus, Lyme disease, linear IgA bullous disease, macular degeneration, megaloblastic anemia, Meniere disease, Mooren's ulcer, mucha-Habermann disease, polymyositis, multiple sclerosis, myasthenia gravis, necrotizing enterocolitis, neuromyelitis optica, ocular pemphigus, ocular myoclonus syndrome, Order thyroiditis, paroxysmal nocturnal hemoglobinuria, Ba-Te two Cotard, pemphigus, periodontitis, pernicious anemia, pollen allergy, polyadenous autoimmune syndromes, posterior uveitis, monolobular cirrhosis, rectitis, pseudomembranous colitis, psoriasis, pulmonary emphysema, pyoderma, Reiter syndrome, reversible obstructive airway disease, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleritis, Sezary syndrome, Sjogren syndrome, subacute bacterium endocarditis, systemic lupus erythematous, Takayasu arteritis, temporal arteritis, painful ophthalmoplegia, type i diabetes, ulcerative colitis, urticaria, vernal conjunctivitis, leukodermia, Wo Ji-Vogt-Koyanagi-Harada syndrome and Wegener granulomatosis.In some embodiments, described illness is endometriosis.
Compound of the present invention also can be used for the medicine preparing treatment animal disorders, and wherein, arrestin kinases can prevent, suppresses or improve symptom or the symptom of described disease.Compound of the present invention also can be used for the medicine preparing treatment or prevention kinase-associated conditions.
Give the mankind belong to the compound shown in formula (I) can by for any accepted pattern (such as oral or per rectum) through enteral administration, or by parenteral admin, such as subcutaneous, intramuscular, intravenously and intradermal route.Injection can be inject or via continue or intermittent infusion.This active compound is contained in pharmaceutically acceptable carrier or thinner usually, and its consumption is enough to treat effective dosage to patient delivery.In various embodiments, compared with normal cell, this inhibitor compound can have fast proliferating cells such as such as tumors to be selected toxicity or has more toxicity.
The compounds of this invention in use, can adopt any bioavailable form of this compound or pattern to give.The stage of the specific nature of the visual selected compounds of technician of preparation formulation art, illness to be treated, illness to be treated and other conditions associated appropriate form and pattern easily selecting administration.We recommend reader with reference to " Lei Mingdun pharmaceutical science ", and the 19th edition, Mack Publishing Company (1995) is to obtain out of Memory.
The compounds of this invention can give separately or give with the form of pharmaceutically acceptable carrier, thinner or vehicle group synthetic drug compositions.Although the compounds of this invention itself is effective, is usually mixed with pharmacy acceptable salt form and gives, because more stable, the easier crystallization and there is higher solubleness usually of these forms.
But these compounds use with the pharmaceutical compositions prepared depending on required mode of administration usually.Therefore, in another embodiment, the invention provides the pharmaceutical composition of compound and pharmaceutically acceptable carrier, thinner or vehicle shown in contained (I).Technology well known in the art can be adopted to prepare these compositions.
In other embodiments, the invention provides a kind of pharmaceutical pack or test kit, it is equipped with the container that one or more is filled with one or more compositions of pharmaceutical composition of the present invention.The container of the medicine containing unitary dose can be housed in this pharmaceutical pack or test kit.These test kits can be equipped with the composition containing active drug, described composition can be enriched material (comprising lyophilised compositions), it first can be done dilution further and re-use the composition that maybe can provide working concentration, and wherein these bottles can be equipped with a or many doses.For the purpose of facility, in test kit, sterile vials can provide single dose, thus make doctor can directly utilize these bottles, wherein the medicine of these small bottle packings expense and concentration to some extent.These containers also can post various written material, the explanation of the governmental agency requirements form of such as working instructions or management medicine or the production of biological product, use or sale, and this explanation reflects that the approval of this mechanism is for the production of human administration, use or sale.
The compounds of this invention can with one or more other drug coupling or administration, to treat described condition/disease.Available same preparation or give all components with different preparation.If with different preparation administration, then the compounds of this invention can give successively or simultaneously with other medicines.
Outside decapacitation is combined with one or more other medicines and given, the compounds of this invention can be used in combination treatment.Now, usual combination with one another gives these compounds.Therefore can simultaneously (as combination preparation) or give one or more the compounds of this invention successively to realize required effect.If the treatment characteristic of each compound is different, especially need like this to make the combined effect of these two kinds of medicines improve treatment result.
Pharmaceutical composition of the present invention for parental injection comprises pharmaceutically acceptable sterile aqueous or non-aqueous solution, dispersion liquid, suspensoid and sterilized powder, thus first can be reconstructed into sterile injectable solution or dispersion liquid re-uses.The example of suitable water-based and non-aqueous carrier, thinner, solvent or mediator comprises the injectable organic esters such as water, ethanol, polyvalent alcohol (such as glycerine, propylene glycol, polyoxyethylene glycol etc.) and suitable mixture, vegetables oil (such as sweet oil) and such as ethyl oleate.By using coating material as Yelkin TTS if dispersion is then by keeping desired particle size and using tensio-active agent, maintain suitable mobility.
These compositions also can comprise auxiliary material, as sanitas, wetting agent, emulsifying agent and dispersion agent.Comprise various antibacterial agent and anti-mycotic agent, such as p-Hydroxybenzoate, butylene-chlorohydrin, phenol, Sorbic Acid etc. are guaranteed to prevent microbial process.Also can need to comprise isotonic agent, such as sugar, sodium-chlor etc.The prolongation that the material postponing to absorb by comprising such as aluminum monostearate and gelatin etc. realizes injectable drug form absorbs.
If need more effective distribution, this compound can be mixed slowly-releasing or targeted delivery systems, as in polymeric matrix, liposome and microsphere.
By, such as filter with bacteria retaining filter or make injectable formulation aseptic by the disinfectant mixing aseptic solid composite form, can before using, by described composition dissolves or be dispersed in sterilized water or other sterile injectable medium.
Solid dosage for oral administration comprises capsule, tablet, pill, pulvis and granule.In these solid dosages, active compound and at least one such as inertia such as Trisodium Citrate or dicalcium phosphate, pharmaceutically acceptable vehicle or carrier and/or following material mix: a) weighting agent or enriching substance, such as starch, lactose, sucrose, glucose, N.F,USP MANNITOL and silicic acid; B) tackiness agent, such as carboxymethyl cellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and gum arabic; C) wetting Agent for Printing Inks, such as glycerine; D) disintegrating agent, such as agar, calcium carbonate, potato or tapioca (flour), alginic acid, some silicate and sodium carbonate; E) solution retarding agents, such as paraffin; F) absorption enhancer, such as quaternary ammonium compound; G) wetting agent, such as hexadecanol and glyceryl monostearate; H) sorbent material, such as kaolin and wilkinite; And i) lubricant, such as talcum powder, calcium stearate, Magnesium Stearate, solid polyethylene glycol, Sodium Lauryl Sulphate BP/USP and their mixture.When capsule, tablet and pill, in formulation, also buffer reagent can be contained.
Also the such as polymkeric substance such as lactose and high molecular weight polyethylene glycol can be used as vehicle, to be filled in soft hard-filled gelatin capsule by solids composition similar for type.
Available dressing and capsid, the casing that such as pharmaceutical-formulating art is known or other dressing are to prepare the solid dosages such as tablet, lozenge, capsule, pill and granule.They optionally can contain opalizer, can also be only or preferentially discharge the composition of active ingredient with delayed mode in certain part (optionally) of enteron aisle.The example of spendable embedding composition comprises polymkeric substance and wax.
If be suitable for, these active compounds and one or more above-mentioned vehicle also can adopt micro-encapsulated form.
The liquid dosage form of oral administration comprises pharmaceutically acceptable emulsion, solution, suspensoid, syrup and elixir.In addition to the active compound, the inert diluent that liquid dosage form can be commonly used containing this area, such as water or other solvent, solubilizing agent and emulsifying agent, such as ethanol, Virahol, ethyl-carbonate, ethyl acetate, benzylalcohol, peruscabin, propylene glycol, 1,3 butylene glycol, dimethyl formamide, oils (especially Oleum Gossypii semen, peanut oil, Semen Maydis oil, germ oil, sweet oil, Viscotrol C and sesame oil), glycerine, tetrahydrofuran (THF) alcohol, polyoxyethylene glycol and sorbitan fatty(acid)ester and their mixture.
Besides inert diluents, these oral compositionss also can comprise the auxiliary reagents such as such as wetting agent, emulsifying and suspending agent, sweeting agent, seasonings and perfume compound.
In addition to the active compound, suspensoid can contain such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitan and sorbitan esters, Microcrystalline Cellulose, the partially suspension agent such as aluminium hydroxide, wilkinite, agar and tragacanth gum and their mixture.
For the preferred suppository of composition of rectum or vagina administration, it is prepared by the suitable non-stimulated vehicle such as mixing the compounds of this invention and such as theobroma oil, polyoxyethylene glycol or suppository wax (they be solid-state in room temperature but under body temperature for liquid, therefore melt and release of active compounds in rectum or vaginal canal) or carrier.
Formulation for the compounds of this invention topical comprises powder agent, patch, sprays, ointment and inhalation.The propellant mixing that active compound aseptically maybe may need with the sanitas of pharmaceutically acceptable carrier and any needs, buffer reagent.
The administered dose of compound preferably can treat and reduce or slow down this illness.Attending doctor adopts routine techniques and is not difficult to determine to treat significant quantity by observing the result obtained under similar state.When determining treatment significant quantity, several factor should be considered, include but not limited to: animal species, volume, age and general health, involved concrete illness, disease serious degree, the reaction of patient for treatment, the particular compound given, mode of administration, give the biological availability of preparation, selected dose therapies, the use of other medicament and other is conditions associated.
Preferred dosage is per kilogram of body weight every day about 0.01 to 300mg scope.Preferred dosage is per kilogram of body weight 0.1 to 100mg scope every day, more preferably every day per kilogram of body weight 0.2 to 80mg, more preferably every day per kilogram of body weight 0.2 to 50mg.Multiple sub-doses can be divided every day to give suitable dosage.
the synthesis of the compounds of this invention
5 step processes shown in employing scheme 1, prepare 5-(9-sec.-propyl-8-methyl-2-morpholine-4-base-9H-purine-6-base)-pyrimidine-2-base amine by dichloropurine.
Scheme 1
Embodiment
In embodiment described below, unless otherwise noted, in below describing all temperature by degree Celsius, all parts and percentage ratio are weight part and weight percentage.
Various parent material and other reagent are buied from chemical suppliers such as such as Aldrich chemical company (AldrichChemicalCompany) or Lancaster synthesis company limited (LancasterSynthesisLtd.), unless otherwise stated, can use without being further purified.Tetrahydrofuran (THF) (THF) and DMF (DMF) are buied in SureSeal bottle from aldrich corp (Aldrich), receive and can use.Unless otherwise noted, otherwise all solvents adopt the standard method purifying of this area.
Below react nitrogen, argon direct draught or use drying tube, carry out in anhydrous solvent in room temperature (unless otherwise indicated), and reaction flask is equipped with rubber septum for introducing material and reagent via syringe.
Glass wares is through oven drying and/or heat drying.Silica gel 60F254 sheet glass (EMerck (0.25mm)) carries out analysis thin-layer chromatography, with appropriate solvent ratio (v/v) wash-out.By TLC analytical reaction, judge reaction terminating by the consumption of parent material.
TLC plate absorbs via UV or uses p-anisaldehyde spraying reagent or Sonnenschein's reagent (Aldrich chemical company, ethanol preparation 20 % by weight) (thermal activation) or dye in iodine room to be observed.
Usually with reaction solvent or extraction solvent, reaction volume is doubled, then use the shown aqueous solution washing of 25% extraction volume (unless otherwise indicated) by volume to carry out aftertreatment.Reaction mixture filters after anhydrous sodium sulfate drying, uses Rotary Evaporators solvent evaporated under reduced pressure, should note during solvent removed in vacuo.
Unless otherwise indicated, otherwise rapid column chromatography (the flashcolumnchromatography) [people such as Still, J.Org.Chem., 43,2923 (1978)] E Merck (Merck) level Flash silica (flashsilica) (47-61mm) is used, silica gel: roughage ratio is about 20:1 to 50:1.Hydrogenolytic cleavage is carried out at shown pressure or under environmental stress.
Bruker instrument is operated with record in 400MHz 1hNMR composes, in 100MHz operating record 13c-NMR composes.Use chloroform as reference standard (7.27ppm and 77.00ppm) or use CD 3oD (3.4 and 4.8ppm and 49.3ppm) as reference standard or suitably time utilize in tetramethylsilane and mark (0.00ppm), obtain CDCl 3nMR spectrum (reporting in units of ppm) of solution.If desired other NMR solvent can be used.When reporting peak diversity, use following abbreviation: s=is unimodal, d=is bimodal, t=triplet, m=multiplet, and br=widens, dd=double doublet, the two triplet of dt=.When providing coupling constant, report in units of hertz.LC/MS is adopted to obtain mass spectrum with ESI or APCI.All fusing points are all not calibrated.The purity of all end products is greater than 90% (via HPLC, 220nm and 254nm wavelength).
Following synthetic example, for illustration of a kind of method of synthesis the compounds of this invention, should not be construed as and is only limitted to these embodiments.
Embodiment 1: synthesis the compounds of this invention
The chloro-9-sec.-propyl of synthesis 2,6-bis--9H-purine
Under room temperature, in 30 minutes, 2,6-dichloropurine (2 mmole), Virahol (8 mmole) and triphenylphosphine (4 mmole) are absorbed in 40 milliliters of anhydrous tetrahydro furans, dropwise add diazonium dicaxboxylate (4 mmole).This reaction mixture at room temperature stirs 24 hours.Periodically this reaction is monitored by TLC or LC/MS.Reaction mixture is poured in the beaker containing ice-cooled water.With 3x100 milliliter extraction into ethyl acetate water layer, obtain crude product.Crude product is chromatography purification (petroleum ether solution of 10-80% ethyl acetate, gradient elution) on a silica gel column, obtains the chloro-9-sec.-propyl of 2,6-bis--9H-purine, productive rate 77%.
Synthesis 5-(the chloro-9-sec.-propyl of 2--9H-purine-6-base)-pyrimidine-2-base amine
To 2, the chloro-9-sec.-propyl of 6-bis--9H-purine (5.21 mmole), 5-(4,4,5,5-tetramethyl--[1,3,2] dioxaborolanes-2-base amine (5.21 mmole) and with 1,1 '-bis-(diphenylphosphino) ferrocene Palladous chloride (II) of methylene dichloride complexing not containing two of superoxide 2M aqueous sodium carbonate (15.6 mmole) is added in alkane (40 milliliters) solution.Reaction mixture is degassed and use nitrogen purging.Then heat in the oil bath of maintenance 80 DEG C and stir this reaction mixture 3 hours.The consumption of initial purine in LC/MS monitoring reaction.
Reaction mixture is cooled to room temperature, and decompression is lower to desolventizing.Resistates is absorbed in the mixture of ethyl acetate and water.Be separated organic phase, water layer 3x100 milliliter ethyl acetate extracts further.Organic phase with sodium sulfate is dry, and solvent removed in vacate, obtains 5-(the chloro-9-sec.-propyl of 2--9H-purine-6-base)-pyrimidine-2-base amine, productive rate 55%.
Synthesis 5-(9-sec.-propyl-2-morpholine-4-base-9H-purine-6-base)-pyrimidine-2-base amine
Morpholine (2.84 mmole) is added in N,N-DIMETHYLACETAMIDE (18 milliliters) solution of 5-(the chloro-9-sec.-propyl of 2--9H-purine-6-base)-pyrimidine-2-base amine (2.84 mmole).Reaction mixture heats and stirs 12 hours in the oil bath of maintenance 94 DEG C.By whether there is not parent material in LC-MS monitoring reaction.Crude product is directly loaded on preparation HPLC post, obtains 5-(9-sec.-propyl-2-morpholine-4-base-9H-purine-6-base)-pyrimidine-2-base amine after chromatogram purification, productive rate 58%. 1HNMR，DMSO-d6:9.53(s，2H)，8.32(s，1H)，7.30(bs，2H)，4.72(m，1H)，3.78(m，4H)，3.73(m，4H)，1.55(d，6H)。m/z:341.17[MH] +.
Synthesis 5-(the bromo-9-sec.-propyl of 8--2-morpholine-4-base-9H-purine-6-base)-pyrimidine-2-base amine
At 5 DEG C, in 15 milliliters of chloroformic solutions of 5-(9-sec.-propyl-2-morpholine-4-base-9H-purine-6-base)-pyrimidine-2-base amine (1.03 grams, 3.03 mmoles), slowly add NBS (594 milligrams, 3.34 mmoles).Continue reaction 2 hours at this temperature.After simple aftertreatment, product 5-(the bromo-9-sec.-propyl of 8--2-morpholine-4-base-9H-purine-6-base)-pyrimidine-2-base amine is by flash column (solvent system: the hexane solution of 50% ethyl acetate), obtain 5-(the bromo-9-sec.-propyl of 8--2-morpholine-4-base-9H-purine-6-base)-pyrimidine-2-base amine, productive rate 52% (660 milligrams). 1HNMR，MeOD:9.67(s，2H)，4.90(m，1H)，3.89(m，4H)，3.82(s，4H)，1.72(d，6H)。m/z:419.31，421.07[MH] +。
Synthesis 5-(9-sec.-propyl-8-methyl-2-morpholine-4-base-9H-purine-6-base)-pyrimidine-2-base amine
In sealed tube, to 5-(the bromo-9-sec.-propyl of 8--2-morpholine-4-base-9H-purine-6-base)-pyrimidine-2-base amine (30 milligrams, 0.072 mmole) and Pd (dppf) Cl 2(3 milligrams, 5% mmole) 3 milliliters anhydrous two in alkane solution, slowly add zinc methide (210 microlitres, 1.0M n-heptane solution).This mixture is heated to about 65 DEG C.Drip MeOH, solvent removed in vacate.In resistates, add EtOAc, the solution 1MHCl of generation, water and salt water washing, then use Na 2sO 4dry.Except desolventizing, crude mixture carries out flash chromatography on silica gel, obtains 5-(9-sec.-propyl-8-methyl-2-morpholine-4-base-9H-purine-6-base)-pyrimidine-2-base amine 8 milligrams, productive rate 47%. 1HNMR，MeOD:9.40(s，2H)，4.81(m，1H)，3.89(m，4H)，3.82(s，4H)，3.71(s，3H)，1.73(d，6H)。m/z:355.16[MH] +。
Embodiment 2: compare bioassay
The more various biological parameter of chemical compound lot the compounds of this invention being synthesized with PCT/SG2008/000379 and discloses.
The parameter of test is:
The activity of TOR test;
The activity of PI3K test;
Solubility test
People's Microsomal Stability is tested
To the method for each test be specifically described below:
mTOR tests
The mTOR kinases of brachymemma and the 4eBP1 inside preparation of His-label.[γ 33p]-ATP from peace agate West Asia (Amersham) (GEHealthcare) buy.Unless otherwise noted, all chemical all derive from company (Sigma-Aldrich) in Sigma-Alder.
Phosphorylation test is carried out at first on the 384 hole polypropylene boards (Greiner) that final volume is 20 microlitres.The test specification of compound is generally 100 μMs to 0.006 μM, point 8 step dilutions, in duplicate.First, in pure DMSO, adding that 10 microlitres/(1.5 mcg/ml mTOR, 40 mcg/ml 4eBP1 are in 1X assay buffer: 10mMHepespH7.5,50mMNaCl and 10mMMnCl for hole 2X enzyme-substrate solution containing in the sample panel of 1 microlitre/hole test compounds 2).By adding 20 μMs of ATP solution in 10 microlitres/hole, (final test concentration is 10 μMs of ATP and 0.4 μ Ci/ hole [γ 33p]-ATP) start reaction.In incubated at room temperature after 1 hour, by the 20mMEDTA/1mMATP solution termination reaction in 40 microlitres/hole.
Then, the mixture of 50 microlitres/hole termination reaction is transferred on 384-hole MultiScreenHTS-PH screen plate (Millipore), this screen plate adds in advance 1% phosphoric acid in 50 microlitres/hole.By vacuum filtration, 0.5% phosphoric acid washing in described plate 120 microlitres/hole four times.Finally, the Optiphase in 10 microlitres/hole is added tMsuperMix liquid scintillation concoction (cocktail) (PerkinElmer).After within minimum 1 hour, cultivating, adopt the coincidence counting pattern of crosstalk correction, count in WallacMicroBetaTriLux scintillation counter.IC 50be defined as the compound concentration needed for 50% maximum possible kinase activity suppression.
pI3K tests
Restructuring PI3Kp110 α/p85 inside preparation.Phosphatidylinositols (PtdIns), phosphatidylserine (PtdSer) and every other unspecified chemical are all purchased from company in Sigma-Alder.[γ 33p] ATP and Optiphase scintillation material can obtain from Pa Jin Elmer Co., Ltd (PerkinElmer).
Test on 384-hole Maxisorp orifice plate (Nunc) with 25 microlitre final test volumes.Compound, according to 3 times of serial dilutions, is tested, usually from 10 μMs with 8 kinds of concentration.Maxisorp orifice plate wraps by the 1:1 mixture of PtdIns and PtdSer in 20 microlitres/hole [being dissolved in chloroform with 0.1 mg/ml separately: in ethanol (3:7)], under room temperature (RT) in stink cupboard dried overnight.
By transfer pipet by 5 microlitres/hole compound (2.5%DMSO solution), 10 microlitres/hole enzyme (0.5 mcg/ml p110 α+1 mcg/ml p85) and 5 μMs, 10 microlitres/hole ATP and 5 μ Ci/ milliliter [γ 33p] ATP adds assay buffer (ultimate density: 0.2 mcg/ml p110 α, 2 μMs of ATP, 0.05 μ Ci/ hole [γ 33p] ATP is in 1X assay buffer: 100mMTris-HClpH7.0,200mMNaCl, 8mMMgCl 2), start enzyme reaction.This reaction at room temperature cultivates 1 hour, by the 50mMEDTA solution termination reaction in 30 microlitres/hole.Then, orifice plate TBS washes twice, and adds the scintillation material in 30 microlitres/hole, then count on MicroBetaTrilux after drying.IC 50be defined as the compound concentration needed for 50% maximum possible kinase activity suppression.
microsomal Stability is tested
In 96 orifice plates (Whatman), adopt the stability of the outer preliminary assessment compound of high-throughput template body, comprise and cultivating with people's liver microsomes (HLM).Verapamil, purchased from company in Sigma-Alder, is used as reference standard in this test.HLM is purchased from Ze Nuo scientific & technical corporation (XenoTech) (20 mg/ml, in 250mM sucrose solution).The mother liquor of previously prepared 100mM potassium phosphate buffer by the following method: by 80 milliliters of 1MK 2hPO 4with 20 milliliters of 1MKH 2pO 4(with dilute hydrochloric acid by pH regulator to 7.4) is mixed, room temperature storage in 900 ml waters.K 2hPO 4.3H 2o and KH 2pO 4purchased from company in Sigma-Alder, NADPH regeneration system rapidly solution A and B are purchased from genetic test company (Gentest).For cancellation reaction stop bath be previously prepared acetonitrile and DMSO (80:20) mixed solution and 4 DEG C of storages.The all solvents used are HPLC levels, and the water used during mother liquor preparation and LC-MS analyze is through the deionization of Milli-Q system.
Mix with 500 microlitre 50mM potassium phosphate buffers (pH7.4, dilute with water 100mM stock buffer dilutes and prepares), the DMSO storing solution of 2.5 microlitre 10mM test compounds is diluted 200 times, obtains 500 microlitre, 50 μMs of solution.Then 8 microlitre compound cocktail are added in the previously prepared cultivation mixed solution that 72 microlitres are made up of water (2250 microlitre), 100mM potassium phosphate buffer (2900 microlitre), NADPH regeneration system rapidly solution B (58 microlitre), NADPH regeneration system rapidly solution A (290 microlitre) and HLM (250 microlitre).Then by the reaction mixture obtained (finalization compound concentration is 5 μMs) in 37 DEG C of incubations in B.BraunCertomatH incubator, then 50 mul aliquots samples are distributed in the hole of the different culture plates comprising 100 ul of stop solution.In 4 DEG C with 2000rpm centrifugal 15 minutes, the sample of 100 μ L gained supernatant liquors is transferred to LC-MS plate and analyzes.Often kind of test compounds samples repeatedly, a series of time point of incubation (5,15,30,45 and 60 minutes).Determine by LC-MS (ABIQtrap3200) concentration remaining compound, compare with the reference solution of concentration known.Then, stability is represented with the transformation period (t1/2, minute).
high-throughput solubility test
Adopt 96-casement plate, the solubleness of deterministic compound in high-throughput dynamic solubility measuring method.By the solubleness of ultraviolet/visible light microplate spectrophotometer (MolecularDevicesSpectraMaxPlus384) assessing compound.Reference standard is used as purchased from Vorinostat (Vorinostat) (SAHA) of Sigma and nicardipine.
Be diluted in the compound in DMSO with phosphate buffered saline buffer (Sigma), ultimate density 250 μMs (5 microlitre 10mM storing solutions are in 195 microliter phosphate buffer pH7) also fully mixes.Then mixture is jolted 1.5 hours with 600rpm, room temperature leaves standstill 2 hours.Then by plate with 1500g centrifugal 15 minutes.Gained supernatant liquor (80 microlitre) be transferred to UV-analysis plates and dilute with DMSO (20 microlitre).Be used in the quantitative sample of calibration mother liquor of the respective compound of preparation in phosphate buffered saline buffer/DMSO (80:20).
The compound of test is as follows:
Table 1 summarizes the result of biological test.
Table 1
As can be seen here, all compounds all have activity to a certain degree, and except compd E (compd E is significantly lower to the activity of mTOR), the compounds of this invention is suitable with the activity of comparative compound to the activity of two kinds of enzymes interested.Therefore, all compounds are all potential drug candidates, although compd E is to the activity of mTOR, some is low.
But, and the compound of not all test all shows acceptable solubility characteristics.Such as, the low solubility result of compd A, B and E shows that these compounds are not desirable drug candidates.Their low solubility makes it be difficult to effectively prepare in acceptable carrier in a physiologically, thus reduces it in human body, has the dynamic (dynamical) possibility of excellent oral pharmaceutical.On the contrary, the solubleness of Compound C, D and the compounds of this invention is acceptable for drug candidate.
But metabolic stability aspect in vitro, result is significantly different.In these tests, compd A, B, E and compound of the present invention have acceptable stability in the research of people's liver microsomes.This is prompting just, if these compounds can successfully give, then they are enough stable to realize required physiological effect in patients.And have at the same time (C, D and compound of the present invention) in the compound of excellent mTOR/PI3K inhibit activities and excellent solubleness, uniquely a kind of compound with acceptable metabolic stability is compound of the present invention.
All compd A-E and compound of the present invention all show excellent activity in these trials, but the activity of the compounds of this invention is the strongest, because it combines potent target spot inhibit activities, excellent water-soluble and excellent metabolic stability.
In a word, the biological results prompting that the compound tested in the above-mentioned biological study carried out realizes, although compound of the present invention and many compare test compounds have obviously close similarity, but the compounds of this invention is unique one meets active, fully water-soluble and metabolic stability requirement, is thus suitable as the compound of drug molecule simultaneously.Therefore, these researchs show that this compound is used as the superiority of drug candidate.
embodiment 3: based on usefulness biomarker test (pp70-S6KT389, pAktS473) of cell
In order to further illustrate the usefulness of the compounds of this invention, two kinds of biomarkers based on cell being carried out to compound of the present invention and tests.Method is as follows:
sureFirep-Akt (Ser473) 384 test kit (TGR, catalog number (Cat.No.): TGRAS500),
sureFirephospho-p70S6 kinases (Thr389) 384 test kit (TGR, catalog number (Cat.No.): TGR70S500) and Proxiplate-384Plus (PerkinElmer, catalog number (Cat.No.): 6008280) purchased from Pa Jin Elmer Co., Ltd (PerkinElmer).PC-3 (PC-3) is purchased from ATCC.Except as otherwise noted, all compounds are all purchased from company in Sigma-Alder.
200 microlitre PC3 cell solutions of 2X105 cells/ml are inoculated in each hole of 96-orifice plate by first day.Inoculate latter 24 hours and add compound, test specification is generally 10 μMs to 4.6nM, point 8 step dilutions, in triplicate.37 DEG C, the ultimate density of 4 hours incubation step period DMSO is 0.1%.After incubation step, removing supernatant liquor, with 1X lysis buffer (AlphaScreen test kit provides) lysing cell and gentleness jolts 10 minutes.The reaction buffer 4 pl of lysis liquid and 5 microlitres being contained AlphaScreen receptor globule adds activation buffer solution mixture and adds each 384-hole (reaction buffer: activate damping fluid: receptor globule is 40:10:1), gentleness jolts 2 hours (room temperature, dark place).2 microlitres are contained the dilution buffer (dilution buffer: donor globule is 20:1) of donor globule adds in each hole of 384-plate, is placed in plate and jolts on device and jolt 1-2 minute, and be incubated overnight at room temperature.
Standard A lphaScreen is adopted to configure (measurement pattern: Alphascreen; Reading mode: Endpoint; Optical mode: AlphaScreen680570; Position postpones: 0.10 second; Firing time: 0.30 second; Integration starts: 0.34 second; Integral time: 0.30 second; Gain: 3000), reads 384-orifice plate with BMGPherostar plate readout instrument.
IC 50be defined as the volumetric molar concentration of the compound needed for kinase activity suppression of 50% maximum possible.5-(9-sec.-propyl-8-methyl-2-morpholine-4-base-9H-purine-6-base)-pyrimidine-2-base amine suppresses p70-S6KT389 and pAktS473phosphorylation iC 50 be respectively 24nM and 9nM.
Biomarker result shows that the compounds of this invention is suppressing the effect in kinase activity.

Claims (35)

1. manufacture a method for the compound of formula (I), the method comprises:
The compound of formula (II) and zinc methide are reacted,
Thus the compound of formula (I) is provided.
2. the method for claim 1, is characterized in that, described reaction is carried out under palladium mixture exists.
3. method as claimed in claim 2, it is characterized in that, described palladium mixture is Pd (dppf) Cl 2.
4. the method for claim 1, is characterized in that, described reaction is carried out in the presence of solvent.
5. method as claimed in claim 4, is characterized in that, described solvent Shi diox.
6. the method for claim 1, is characterized in that, described reaction is carried out in sealed tube.
7. the method for claim 1, is characterized in that, described reaction is at high temperature carried out.
8. method as claimed in claim 7, it is characterized in that, described temperature is about 65 DEG C.
9. manufacture a method for the compound of formula (II), the method comprises:
The compound of formula (III) and N-bromosuccinimide are reacted,
Thus the compound of formula (II) is provided.
10. method as claimed in claim 9, it is characterized in that, described reaction is carried out in the presence of solvent.
11. methods as claimed in claim 10, it is characterized in that, described solvent is chloroform.
12. methods as claimed in claim 9, it is characterized in that, described reaction is carried out at low temperatures.
13. methods as claimed in claim 12, is characterized in that, described temperature is about 5 DEG C.
The method of the compound of 14. 1 kinds of manufactures formula (III), the method comprises:
The compound of formula (IV) and morpholine are reacted,
Thus the compound of formula (III) is provided.
15. methods as claimed in claim 14, it is characterized in that, described reaction is carried out in the presence of solvent.
16. methods as claimed in claim 15, it is characterized in that, described solvent is N,N-DIMETHYLACETAMIDE.
17. methods as claimed in claim 14, it is characterized in that, described reaction is at high temperature carried out.
18. methods as claimed in claim 17, is characterized in that, described temperature is about 94 DEG C.
The method of the compound of 19. 1 kinds of manufactures formula (IV), the method comprises:
The compound of the compound of formula (V) and formula (VI) is reacted,
Thus the compound of formula (IV) is provided.
20. methods as claimed in claim 19, it is characterized in that, described reaction is carried out in the presence of solvent.
21. methods as claimed in claim 20, it is characterized in that, described solvent is methylene dichloride.
22. methods as claimed in claim 19, is characterized in that, described reaction is carried out under palladium mixture exists.
23. methods as claimed in claim 22, it is characterized in that, described palladium mixture is 1,1 '-bis-(diphenylphosphino) ferrocene palladium (II) muriate.
24. methods as claimed in claim 19, it is characterized in that, described reaction is carried out in the presence of a base.
25. methods as claimed in claim 24, it is characterized in that, described alkali is sodium carbonate.
26. methods as claimed in claim 19, it is characterized in that, described reaction is at high temperature carried out.
27. methods as claimed in claim 26, is characterized in that, described temperature is about 80 DEG C.
28. method as claimed in claim 19, it is characterized in that, the compound of described formula (IV) is by purification by liquid extraction.
The application of compound in the medicine for the preparation for the treatment of Animal diseases of the formula (I) of 29. claims 1, wherein, one or more protein kinases being selected from serine/threonine protein kitase or its fragment or mixture or PI3 kinases or its fragment or mixture are suppressed to prevent, suppress or improve symptom or the symptom of described disease.
30. application as described in claim 29, it is characterized in that, described illness is cancer.
31. apply as claimed in claim 30, it is characterized in that, described cancer is selected from lower group: hematologic cancer, myeloproliferative disease, idiopathic myelofibrosis, polycythemia vera, essential thrombocythemia, chronic myeloid leukemia, myeloid metaplasia, chronic myelomonocytic leukemia, acute lymphoblastic leukemia, acute erythroblastic leukemia, Huo Qijin and non-Hodgkin lymphoma, B cell lymphoma, acute T-cell leukemia, myelodysplastic syndrome, plasmocyte obstacle, hairy cell, Kaposi sarcoma, lymphoma, and hyperproliferation illness, psoriasis and restenosis, gynecological cancer, mammary cancer, ovarian cancer, cervical cancer, vagina and vulva cancer, endometrial hyperplasia, gastrointestinal cancer, colorectal carcinoma, polyp, liver cancer, cancer of the stomach, carcinoma of the pancreas, carcinoma of gallbladder, urinary tract cancer, prostate cancer, kidney and kidney portion cancer, bladder cancer, urethral carcinoma, penile cancer, skin carcinoma, melanoma, cerebral tumor, glioblastoma, neuroblastoma, astrocytoma, ependymoma, brain stem glioma, medulloblastoma, meningioma, astrocytoma, oligodendroglioma, incidence cancer, nasopharyngeal carcinoma, laryngocarcinoma, respiratory cancer, lung cancer, NSCLC and SCLC, mesothelioma, illness in eye, retinoblastoma, musculoskeletal disease, osteosarcoma, flesh and bone tumor, squamous cell cancer and fibroma.
32. apply as claimed in claim 29, it is characterized in that, described illness is precancerosis disease or hyperplasia.
33. apply as claimed in claim 32, it is characterized in that, described illness is selected from: tumorigenesis in familial adenomatous polyposis, adenomatous polyposis coli, myelodysplasia, Endometrium development exception, endometrial hyperplasia with atypia, cervical atypical hyperplasia, vagina epithelium, benign prostatic hyperplasia, larynx papilloma, photochemical and solar keratosis, seborrheic keratosis and keratoacanthoma.
34. apply as claimed in claim 29, it is characterized in that, the disease that described illness is autoimmunization or inflammatory diseases or is caused by excessive neovascularization.
35. apply as claimed in claim 34, it is characterized in that, described illness is selected from: acute disseminated encephalomyelitis, addison's disease, without gamma-globulin mass formed by blood stasis, granulopenia, allergic asthma, allergic encephalitis, rhinallergosis, alopecia areata, senile alopecia, red corpuscle occurs can not, ankylosing spondylitis, antiphospholipid antibody syndrome, aortitis syndrome, aplastic anemia, atopic dermatitis, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmunization ovaritis, balo disease, Basedow disease, behcet disease/syndrome, bronchial asthma, block this Mann syndrome, celiac disease, chagas disease, chronic inflammatory demyelinating polyneuropathy, churg-Strauss syndrome, Ke's radicular syndrome, keratoconus, keratoconus hickie, coxsackie myocarditis, CREST is sick, Crohn's disease, epidermis eosinophilia, cutaneous T-cell lymphoma, diversity dermatitis erythremia, dermatomyositis, diabetic retinopathy, postmyocardial infarction syndrome, dystrophia epithelialis corneae, eczematoid dermatitis, eosinophilic fasciitis, eosinophilic gastroenteritis, epidermolysis bullosa, Evans syndrome, FA, gestation pemphigoid, glomerulonephritis, Goodpasture's syndrome, graft versus host disease (GVH disease), Graves disease, guillain-Barre syndrome, Hashimoto's disease, hemolytic uremic syndrome, herpetic keratitis, ichthyosis vulgaris, idiopathic interstitial pneumonia, idiopathic thrombocytopenic purpura, inflammatory bowel, kawasaki disease, keratitis, keratoconjunctivitis, Lambert-Eaton syndrome, common white pinta, lichen planus, lichen sclerosus, Lyme disease, linear IgA bullous disease, macular degeneration, megaloblastic anemia, Meniere disease, Mooren's ulcer, mucha-Habermann disease, polymyositis, multiple sclerosis, myasthenia gravis, necrotizing enterocolitis, neuromyelitis optica, ocular pemphigus, ocular myoclonus syndrome, Ao Deshi thyroiditis, paroxysmal nocturnal hemoglobinuria, Ba-Te two Cotard, pemphigus, periodontitis, pernicious anemia, pollen allergy, polyadenous autoimmune syndromes, posterior uveitis, monolobular cirrhosis, rectitis, pseudomembranous colitis, psoriasis, pulmonary emphysema, pyoderma, Reiter syndrome, reversible obstructive airway disease, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleritis, Sezary syndrome, Sjogren syndrome, subacute bacterium endocarditis, systemic lupus erythematous, Takayasu arteritis, temporal arteritis, painful ophthalmoplegia, type i diabetes, ulcerative colitis, urticaria, vernal conjunctivitis, leukodermia, Wo Ji-Vogt-Koyanagi-Harada syndrome and Wegener granulomatosis.
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CN1426398A (en) * 2000-04-27 2003-06-25 山之内制药株式会社 Condensed heteroaryl derivatives
US20040176385A1 (en) * 2002-11-21 2004-09-09 Nuss John N. Small molecule PI 3-kinase inhibitors and methods of their use
GB2431156A (en) * 2005-10-11 2007-04-18 Piramed Ltd 1-cyclyl-3-substituted- -benzenes and -azines as inhibitors of phosphatidylinositol 3-kinase
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